Abstract

The receptor tyrosine kinase erbB2 is known to play a role in the development of hepatic progenitor cells and is often found to be expressed and mutated in different solid tumors. Activated erbB2 is a marker of poor prognosis in several malignancies and to date, successful strategies have been developed to target erbb2 receptor. The aim of this study was to determine the expression of erbb2 in human hepatoblastoma, the most common primary hepatic malignancy in early childhood, and its functional role in hepatoblastoma cell biology. Expression of erbB2 on the protein level was analyzed by Western Blot, immunofluorescence and RT-PCR of lysates of primary tumors and cell lines HuH6, HepT1, HepT3 and HepG2. Immunohistochemical staining was performed to determine phosphorylated erbB2 and subsequent kinases. Single strand conformation polymorphism (SSCP) analysis was used to scan known hotspots for mutations (exons 6, 8, 18–23 of erbB2) in a panel of 56 tumor samples. Cells were stimulated with rhHRG1-β. Activation of subsequent kinases was determined via Western Blot and MTT assay was used to determine proliferation after stimulation and overexpression of erbb2 via transient transfection. The influence of rhHRG1-β on the activity of the Wnt/ß-catenin pathway was measured by transfection with the TOP-/FOP-Flash reporter assay. The effects of erbB2 kinase knockdown by transient transfection with siRNA against erbB2 on apoptosis and proliferation were also evaluated. Expression of erbB2 was detected in all four cell lines and most of the primary tumors. Immunohistochemistry indicated expression of phosphorylated (activated) Erbb2 proteins in 90% and activation of subsequent kinases, which are downstream targets of erbb2. Mutations were not present in the erbB2 gene. Stimulation with HRG1-β induced phosphorylation of Akt, Erk1/2 and p70S6K and resulted in increased cell proliferation. This effect was not mediated by an increase of activity of the Wnt/ß-catenin signaling pathway because TOP-Flash reporter activity was not further increased in the presence of rhHRG1-β. Overexpression of erbB2 resulted in enhanced proliferation after stimulation with HRG1-β in all four cell lines, whereas knockdown of erbB2 decreased the proliferative effect of HRG1-β on hepatoblastoma cell lines. Knockdown of erbB2 did not induce apoptosis in hepatoblastoma cell lines. These results indicate that erbB2 might play an important functional role in hepatoblastoma cell biology. Further experiments are needed to evaluate the clinical significance.